Modem electron microscopy of biological specimens is more and more frequently examining specimens that have been immobilized by freezing (cryofixation). Freezing must be performed in such a way that the specimens vitrify, i.e. so that upon cooling, no ice (which would segregate the specimen) can form. “Segregation” is the separation of the specimen into pure water (ice crystals) and concentrated dissolved substances between the branches of the ice crystals. In vitrification, water is immobilized in its liquid configuration. In other words, all the constituents of the biological specimen are not displaced during immobilization, and thus represent a “snapshot” of the living material at the moment it was fixed. Vitrification in a biological system is a rapid process: a freezing rate of several 100,000 K/s is normally necessary. For physical reasons, such high freezing rates can be achieved only in very thin specimens (thickness <20 μm). The critical freezing rate necessary for vitrification can be reduced by a factor of 100 if the specimen is frozen at a pressure of approximately 2000 bar. In so-called high-pressure freezing (Studer et al., Journal of Microscopy 179 (1995), 321–332), biological specimens approx. 200 μm thick are vitrified. Production of a 200 μm-thick specimen with a diameter of 1.2 to 3 mm is difficult. The advantages of high-pressure freezing are reduced if a great deal of time is spent preparing the biological specimens, e.g. a piece of tissue, for freezing. Individual attempts at rapid preparation of very small tissue pieces have been described by various authors (H. Hohenberg et al., Journal of Microscopy 183 (1996), 133–139; E. Shimoni et al., Journal of Microscopy 192 (1998), 236–247). Shimoni et al. fired small metal capillaries (gold tubules with an inside diameter of 200 μm) into the tissue; locating these capillaries in the tissue is time-consuming, however, and the frozen specimens are difficult to process. Hohenberg et al. (1996) took tissue samples 0.2–0.25×0.4×4–8 mm in size from experimental animals, using commercially available microbiopsy needles adapted for that particular purpose.
Commercially obtainable apparatuses for taking microbiopsy samples from living tissue have a spring mechanism (gun) that receives a needle (for example, Pro-Mag™, Manan Medical Products, Inc., Northbrook, Ill. 60062, USA). This biopsy needle in turn comprises a solid lance (diameter 0.6–3 mm) having an opening, 8–16 mm long and 0.3–2 mm deep, for receiving the biopsy material (Hohenberg op. cit. FIG. 1-D, pp. 134–35). This lance is pointed at one end, and has a holding device at the other end. This lance is in turn surrounded by a thin-walled hollow needle whose inside diameter corresponds to the outside diameter of the lance, so that the lance and hollow needle are displaceable with respect to one another. The hollow needle has a cutting edge at one end and a holding device at the other.
The hollow needle, and the lance inserted in it, are secured with the holding devices in the gun, so that the mechanism for removal of the biopsy material is as follows: First the needle, inserted into the gun, is introduced into the tissue, so that the hollow needle allows only the tip of the lance to protrude. Then the spring mechanism is released. As a result, firstly the lance is advanced farther into the tissue so that the opening is no longer enclosed by the hollow needle. The tissue enters the opening, and at the next instant the hollow needle with the cutting edge is in turn slid over the opening by the spring mechanism. The piece of tissue present in the opening is thereby cut off. The hollow needle, together with the lance, is then pulled back out of the tissue, and the outer hollow needle is once again slid back with respect to the lance so that the cut-out specimen becomes accessible.
Biopsy specimens obtained in this fashion have, for example, the following dimensions: 200 to 250 μm high, 400 μm wide, and 4 to 8 mm long (Hohenberg, op. cit., p. 135). These specimens are manually reduced in size using suitable cutting tools (razor blades, scalpels, or the like), cut to the correct size, and transferred with forceps or other suitable instruments into the specimen plates of appropriate high-pressure freezing units.
This manual preparation of biopsy specimens for high-pressure freezing exhibits, however, a number of serious disadvantages which reveal this method to be unsuitable for larger quantities of specimens and especially for routine operation. The elapsed time between excision of the biopsy material and the beginning of the freezing operation has been quoted as approx. 40 seconds (Hohenberg op. cit. p. 135); experience has shown, however, that this is a very optimistic time scale. This elapsed time is obviously too great, particularly in cases in which “snapshots” need to be prepared of cells whose ultrastructure changes rapidly, for example muscle cells. A further disadvantage of manual manipulation of biopsy specimens is the fact that the gelatinous consistency and small dimensions of the tissue specimens considerably complicate reliable manual manipulation thereof, so that the reproducibility of the results is low and the reject rate is considerable. This in turn not only is undesirable for general cost reasons, but also is unacceptable especially in situations in which consideration must be given to the patient/examination subject or experimental animal, and excision of biopsy material therefore cannot be repeated an unlimited number of times, for example in the case of removal of specimens from a living myocardium. Lastly, the most important disadvantage of manual manipulation of biopsy specimens is the fact that this manipulation is so difficult to perform that, as indicated by experience, only a small percentage of the technical personnel of an institution are at all capable, even after extended training, of producing perfect or at least satisfactory specimens. The results of manual preparation are therefore poorly reproducible, and the user of commercial equipment must resort to employing, for preparation, persons who by fortunate accident possess manual and motor skills far above the average.